Process for the preparation of D-pantolactone

ABSTRACT

A process for the preparation of D-pantolactone, comprising employing specific microorganisms to convert D,L-pantolactone as the starting material into D-pantoic acid by selective asymmetric hydrolysis of the D-form only in the D,L-pantolactone, and then separating D-pantoic acid and converting it into D-pantolactone.

FIELD OF INDUSTRIAL APPLICATION

The present invention relates to a process for the preparation ofD-pantolactone, a useful intermediate in the preparation ofD-pantothenic acid and pantethine, both useful as vitamins of medical orphysiological importance.

PRIOR ART

D-pantolactone has heretofore been prepared through optical resolutionof chemically synthesized D,L-pantolactone.

Such process, however, requires the use of costly resolving agents suchas quinine or brucine, and has the disadvantage that the recovery ofD-pantolactone is not easily carried out.

Processes of enzymatically resolving D,L-pantolactone are also known,and the following processes have heretofore been reported:

In Japanese Examined Patent Application Publication No. 19745/72(TOKKO-SHO 47-19745) is described a process of obtaining onlyD-pantolactone by using microorganisms to completely decomposeL-pantolactone in D,L-pantolactone. This process, however, has thedrawback that half the amount of D,L-lactone is lost.

In Japanese Unexamined Patent Application Publication No. 293386/86(TOKKAI-SHO 61-293386) is described a process wherein onlyL-pantolactone in D,L-pantolactone is oxidized by the use ofmicroorganisms into ketopantolactone, which is then converted byasymmetric reduction into D-pantolactone. This process, however, is oflittle practical significance due to the fact that both the substrateconcentration and the reaction rate are low.

In Japanese Unexamined Patent Application Publication Nos. 152895/82(TOKKAI-SHO 57-152895) and 294092/87 (TOKKAI-SHO 62-294092) aredescribed processes wherein the L-form in D,L-pantolactone isselectively subjected to asymmetric hydrolysis by microorganisms toafford D-pantolactone. These processes are not practical because boththe substrate concentration and the reaction rate are low, andD-pantolactone of high optical purity can be obtained only when theL-form has been completely hydrolyzed.

DISCLOSURE OF THE INVENTION

As a result of extensive researches on the asymmetric hydrolysis ofD,L-pantolactone, the present inventors have now found thatD-pantolactone can be obtained efficiently from D,L-pantolactone throughselective asymmetric hydrolysis by certain microorganisms of only theD-form in D,L-pantolactone to form D-pantoic acid, followed byseparation and conversion thereof into D-pantolactone. The presentinvention has been predicated on such findings.

Accordingly, the present invention provides a process for thepreparation of D-pantolactone, comprising selectively subjecting theD-form in D,L-pantolactone to asymmetric hydrolysis employing amicroorganism possessing the ability to effect said selective asymmetrichydrolysis selected from the group consisting of the genera Fusarium,Cylindrocarpon, Gibberella, Aspergillus, Penicillium, Rhizopus,Volutella, Gliocladium, Eurotium, Nectria, Schizophyllum, Myrothecium,Neurospora, Acremonium, Tuberculina, Absidia, Sporothrix, Verticilliumand Arthroderma to form D-pantoic acid, which is then separated andconverted into D-pantolactone, followed by recovery thereof.

As compared to the above-mentioned known processes of selectiveasymmetric hydrolysis of the L-form in D,L-pantolactone, the presentinvention has many advantages. For example higher substrateconcentrations may be used, shorter reaction times may be employed, andD-pantolactone of extremely high optical purity can be obtained.

The following describes the present invention in more detail.

The inventors inoculated 5 ml portions of different liquid media withseed cultures from slants. The seeded media were subjected to aerobicshake culture at 28° C. for 2-7 days and then to centrifugation orfiltration to collect the cells. To the cells were added 2 ml of 2%D,L-pantolactone solution in 0.2M Tris-HCl buffer, and the mixture wasshaken overnight at 28° C. The resultant reaction liquid was subjectedto HPLC and GLC to measure the decrease of pantolactone and the amountof pantoic acid formed, and to determine the optical purity ofpantolactone, respectively.

As a result thereof, it has been found that microorganisms with theability to carry out asymmetric hydrolysis which belong to the generaFusarium, Cylindrocarpon, Gibberella, Aspergillus, Penicillium,Rhizopus, Volutella, Gliocladium, Eurotium, Nectria, Schizophyllum,Myrothecium, Neurospora, Acremonium, Tuberculina, Absidia, Sporothrix,Verticillium or Arthroderma have suitable properties for theD-form-selective asymmetric hydrolysis and for the industrial productionof D-pantolactone.

Among microorganisms belonging to each of the genera mentioned above canbe found those that exhibit a particularly outstanding ability to carryout the D-form-selective asymmetric hydrolysis.

In the process according to the present invention, conditions underwhich to cultivate the microorganisms will vary with the strain used.With regard to media, there are used such media which containsaccharides such as glucose or sucrose, alcohols such as ethanol orglycerol, fatty acids such as oleic acid, or stearic acid or estersthereof, or oils such as rapeseed oil or soybean oil as carbon sources;ammonium sulfate, sodium nitrate, peptone, Casamino acids, corn steepliquor, bran, yeast extract or the like as nitrogen sources; magnesiumsulfate, sodium chloride, calcium carbonate, potassium monohydrogenphosphate, potassium dihydrogen phosphate or the like as inorganic saltsources; and malt extract, meat extract or the like as other nutrientsources. The cultivation is carried out aerobically, normally for anincubation period of 1-7 days at a medium pH of 3-9 and an incubationtemperature of 10°-50° C.

Microorganisms to be used in the process of the present invention may bein any form, for example, cultures obtained by cultivation of strains inliquid media, cells separated from liquid cultures, dried cells obtainedby processing cells or cultures, or immobilized cells.

The operation may be carried out batchwise, semi-batchwise orcontinuously. The concentration of D,L-pantolactone used is normally10-500 g/l. The reaction temperature is normally 10°-50° C. and thereaction time, in the case of batchwise operation, is normally severalhours to three days. The pH of the reaction system is normally of theorder of 3-8.

As a result of the D-form-selective asymmetric hydrolysis ofD,L-pantolactone by microorganisms, D-pantoic acid is formed with the pHof the reaction liquid being lowered and the reaction rate beingdecreased concomitantly. In order to maintain higher reaction rates itis desirable for the reaction liquid to be maintained at a pH optimalfor the particular microorganism's lactone-hydrolyzing enzyme. For thispurpose, hydroxides or carbonates of alkaline or alkaline earth metals,as well as aqueous ammonia or the like, are used as inorganic bases withwhich to maintain pH.

After the reaction has been completed, L-pantolactone in the reactionliquid which has not undergone hydrolysis is separated, for examplethrough extraction with organic solvents. D-pantoic acid remaining inthe reaction liquid is then heated under acidic conditions forconversion into D-pantolactone. The resultant D-pantolactone isrecovered by extraction with organic solvents. The recoveredL-pantolactone is racemized in any conventional manner for conversioninto D,L-pantolactone. This D,L-pantolactone may be used anew byrecycling it as the starting material for the process according to thepresent invention.

The following examples are given to illustrate the present inventionmore specifically, but the invention is in no way restricted to theseexamples.

EXAMPLE NOS. 1-19

A liquid medium (pH 6.5) consisting of 1% glucose, 0.5% peptone, 0.5%yeast extract and 0.5% corn steep liquor was dispensed in 5 ml portionsinto test tubes, and then heat-sterilized by autoclaving at 121° C. for20 minutes. The various strains mentioned in Table 1 were eachinoculated from slants into the medium in the test tubes, and subjectedto aerobic shake culture at 28° C. for 5 days. After the cultivation,cells were collected by filtration. Into containers each containingdifferent collected cells was dispensed a 2% D,L-pantolactone solutionin Tris-HCl buffer (pH 7.5) in 2 ml portions, and the containers wereshaken overnight at 28° C. After the reaction, cells were removed byfiltration, and each reaction liquid was subjected to HPLC (Nucleosil5C₁₈ φ 4.6×l 150 mm; eluent 10% methanol; rate of flow 1 ml/min;wavelength for detection 230 nm) to determine the decrease inpantolactone and the amount of pantoic acid formed. Unreactedpantolactone in the reaction liquid is separated by extraction withethyl acetate, and pantoic acid remaining in the reaction liquid is thenheated under acidification with hydrochloric acid for lactonization. Theresultant D-pantolactone was extracted with ethyl acetate. The opticalpurity of the D-pantolactone thus obtained from the pantoic acid wasmeasured by GLC (Analytical Biochemistry 112, 9-19 (1981)). The resultsare as shown in Table 1.

                  TABLE 1                                                         ______________________________________                                                                         Optical                                                              Rate of  Purity of                                    Example                                                                              Identification   Hydroly- D-pantolactone                               No.    of the Strain    sis in % in % e.e.                                    ______________________________________                                         1     Fusarium oxysporum                                                                             30.1     91.2                                                IFO 5942                                                                2     Cylindrocarpon tonkinense                                                                      25.4     95.1                                                IFO 30561                                                               3     Gibberella fujikuroi                                                                           27.2     93.7                                                IFO 6349                                                                4     Aspergillus awamori                                                                            13.8     81.7                                                IFO 4033                                                                5     Penicillium chrysogenum                                                                        30.9     79.3                                                IFO 4626                                                                6     Rhizopus oryzae  15.4     77.1                                                IFO 4706                                                                7     Volutella buxi    9.0     77.4                                                IFO 6003                                                                8     Gliocladium catenulatum                                                                         4.0     72.1                                                IFO 6121                                                                9     Eurotium chevalieri                                                                            30.0     67.2                                                IFO 4334                                                               10     Nectria elegans  12.3     75.0                                                IFO 7187                                                               11     Schizophyllum commune                                                                          18.5     76.5                                                IFO 4928                                                               12     Myrothecium roridum                                                                             9.2     64.3                                                IFO 9531                                                               13     Neurospora crassa                                                                              14.5     42.5                                                IFO 6067                                                               14     Acremonium fusidioides                                                                         23.3     49.2                                                IFO 6813                                                               15     Tuberculina persicina                                                                           9.6     40.1                                                IFO 6464                                                               16     Absidia lichtheimi                                                                             22.2     34.8                                                IFO 4009                                                               17     Sporothrix schenckii                                                                           11.6     32.4                                                IFO 5983                                                               18     Verticillium malthousei                                                                         9.1     49.6                                                IFO 6624                                                               19     Arthroderma uncinatum                                                                          10.8     34.3                                                IFO 7865                                                               ______________________________________                                         N.B.: IFO No. stands for No. in the Catalog issued by ZAIDANHOJIN             HAKKOKENKYU-SHO (Institute for Fermentation Osaka, a juridical foundation                                                                              

EXAMPLE NOS. 20-23

Using 500 ml shake flasks each containing 100 ml of a liquid medium (pH5.5) consisting of 2% glycerol, 0.5% peptone, 0.5% yeast extract and0.5% corn steep liquor, the different strains mentioned in Table 2 wereeach subjected to aerobic shake culture at 28° C. for 6 days. After thecultivation, the different cells were collected by filtration and placedseparately in containers. To these containers were added 25 ml portionsof a 30% aqueous D,L-pantolactone solution. The reaction liquid was keptat a pH of 6.5- 7.5 while adding dropwise 28% aqueous ammonia withstirring, and the reaction was allowed to proceed at 28° C. for 2 days.After-treatment was carried out in the same manner as in Example Nos.1-19. The yields in terms of amount and percentage and [α]_(D) ²⁰ areshown in Table 2 for D-pantolactone obtained and L-pantolactonerecovered.

                  TABLE 2                                                         ______________________________________                                        Ex-  Identi- D-Pantolactone formed                                                                         L-Pantolactone formed                            am-  fication                                                                              Yield-  Yield-                                                                              [α].sub.D .sup.20                                                             Yield-                                                                              Yield-                                                                              [α].sub.D .sup.20          ple  of the  ing     ing   (c = 2,                                                                             ing   ing   (c = 2,                          No.  strain  in g    in %  Water)                                                                              in g  in %  Water)                           ______________________________________                                        20   Fusar-  2.76    36.8  -45.6°                                                                       4.42  58.9  +29.4°                         ium                                                                           oxy-                                                                          sporum                                                                        IFO                                                                           5942                                                                     21   Fusar-  2.66    35.5  -44.1°                                                                       4.19  55.9  +24.6°                         ium                                                                           semi-                                                                         tectum                                                                        IFO                                                                           30200                                                                    22   Cylin-  1.70    22.7  -45.1°                                                                       5.16  68.8  +13.6°                         drocar-                                                                       pon                                                                           tonkin-                                                                       ense                                                                          IFO                                                                           30561                                                                    23   Gibber- 2.53    33.7  -44.3°                                                                       4.44  59.2  +23.8°                         ella                                                                          fuji-                                                                         kuroi                                                                         IFO                                                                           6349                                                                     ______________________________________                                         N.B.: IFO No. stands for No. in the Catalog issued by ZAIDANHOJIN             HAKKOKENKYU-SHO (Institute for Fermentation Osaka, a juridical foundation

We claim:
 1. A process for preparing D-pantolactone, comprisingsubjecting D,L-pantolactone to selective asymmetric hydrolysis bycontacting said D,L-pantolactone with a microorganism possessing theability to effect selective asymmetric hydrolysis of only D-pantolactonein said D,L-pantolactone selected from the group consisting of thegenera Fusarium, Cylindrocarpon, Gibberella, Aspergillus, Volutella, andGliocladium to produce D-pantoic acid, and then separating saidD-pantoic acid from the reaction medium, converting it intoD-pantolactone, and recovering said D-pantolactone,
 2. The process ofclaim 1, wherein said microorganism is grown aerobically in a medium fora period of 1 to 7 days, at a pH of 3 to 9, at a temperature of 10° to50° C.
 3. The process of claim 2, wherein said carbon source is selectedfrom the group consisting of a saccharide, an alcohol, a fatty acid, afatty acid ester, and an oil.
 4. The process of claim 3, wherein saidsaccharide is selected from the group consisting of glucose and sucrose,said alcohol is selected from the group consisting of ethanol andglycerol, said fatty acid is selected from the group consisting of oleicacid and stearic acid, said fatty acid ester is selected from the groupconsisting of an ester of oleic acid and an ester of stearic acid, andsaid oil is selected from the group consisting of rapeseed oil andsoybean oil; said nitrogen source is at least one member selected fromthe group consisting of ammonium sulfate, sodium nitrate, peptone,Casamino acids, corn steep liquor, bran and yeast extract; and saidother nutrient source is at least one member selected from the groupconsisting of malt extract and meat extract.
 5. The process of claim 1,wherein said microorganism is in a form selected from the groupconsisting of a culture obtained by cultivation of a strain thereof in aliquid medium, cells separated from a liquid culture, dried cellsobtained by processing cells or cultures, and immobilized cells.
 6. Theprocess of claim 1, wherein said process is carried out batchwise,semi-batchwise, or continuously.
 7. The process of claim 1, wherein theconcentration of D,L-pantolactone in said reaction medium is initiallyfrom 10 to 500 g/l.
 8. The process of claim 6, wherein said batchwiseprocess is carried out for a time in the range of from several hours tothree days.
 9. The process of claim 2, wherein the pH of the reactionsystem is in the range from 3 to
 8. 10. The process of claim 1, whereinthe reaction system is maintained at a pH optimal for the activity ofthe enzyme responsible for catalyzing said selective asymmetrichydrolysis.
 11. The process of claim 10, wherein said optimal pH ismaintained by employing an inorganic base.
 12. The process of claim 11,wherein said inorganic base is selected from the group consisting of ahydroxide of an alkaline metal, a hydroxide of an alkaline earth metal,a carbonate of an alkaline metal, a carbonate of an alkaline earthmetal, and aqueous ammonia.
 13. The process of claim 1, whereinunreacted L-pantolactone remaining in the reaction medium is removed viaextraction with a first organic solvent, the D-pantoic acid remaining inthe reaction medium is then heated under acidic conditions to convert itto D-pantolactone, and said D-pantolactone is recovered via extractionwith a second organic solvent.
 14. The process of claim 13, wherein saidL-pantolactone is racemized to produce D,L-pantolactone.
 15. The processof claim 13, wherein said first organic solvent is ethyl acetate andsaid second organic solvent is ethyl acetate.
 16. A process forpreparing D-pantolactone, comprising subjecting D,L-pantolactone toselective asymmetric hydrolysis by contacting said D,L-pantolactone witha microorganism possessing the ability to effect selective asymmetrichydrolysis of only D-pantolactone in said D,L-pantolactone selected fromthe group consisting ofAbsidia lichtheimi IFO 4009, Acremoniumfusidioides IFO 6813, Arthroderma uncinatum IFO 7865 Aspergillus awamoriIFO 4033, Cylindrocarpon tonkinense IFO 30561, Eurotium chevalieri IFO4334, Fusarium oxysporum IFO 5942, Fusarium semitectum IFO 30200,Gibberella fujikuroi IFO 6349, Gliocladium cantenulatum IFO 6121,Myrothecium roridum IFO 9531, Nectria elegans IFO 7187, Neurosporacrassa IFO 6067, Penicillium chrysogenum IFO 4626, Rhizopus oryzae IFO4706, Schizophyllum commune IFO 4928, Sporothrix schenckii IFO 5983,Tuberculina persicinia IFO 6464, Verticillium malthousei IFO 6624, andVolutella buxi IFO 6003to produce D-pantoic acid, and then separatingsaid D-pantoic acid from the reaction medium, converting it intoD-pantolactone, and recovering said D-pantolactone.
 17. A process forpreparing D-pantolactone, comprising subjecting D,L-pantolactone toselective asymmetric hydrolysis by contacting said D,L-pantolactone witha microorganism possessing the ability to effect selective asymmetrichydrolysis of only D-pantolactone in said D,L-pantolactone selected fromthe group consisting of the genera Fusarium, Cylindrocarpon, Gibberella,Aspergillus, Volutella, and Gliocladium to produce D-pantoic acid, andthen separating said D-pantoic acid from the reaction medium, convertingsaid D-pantoic acid into D-pantolactone, and recovering saidD-pantolactone, wherein said microorganism is grown aerobically in amedium for a period of 1 to 7 days, at a pH of 3 to 9, at a temperatureof 10° to 50° C.